Chapter 3. Frequency-Dependent Synaptic Transmission and Plasticity by
3.3 Results
3.3.1 DA selectively depresses excitatory synaptic transmission at TA-CA1 pyramidal neuron synapses
To examine the differential influence of DA on the two excitatory inputs to area CA1, we made extracellular field recordings from both the Schaffer-collateral (SC) pathway and the temporoammonic (TA) pathway in hippocampal slices (Figure 3-1A). As previously described (Otmakhova and Lisman, 1999), when DA (20 µM) was applied to the bathing solution, the field EPSP (fEPSP) evoked by the TA pathway stimulation was depressed, whereas the fEPSP by the SC pathway stimulation was not significantly altered (Figure 3-1B; DA: 49.2 ± 8.8%, SC: 93.6 ± 15.8%, mean percentage of baseline 20 – 30 min after DA application). To identify the synaptic locus of DA’s effect, we conducted whole-cell voltage-clamp recordings from CA1 pyramidal neurons. DA also depressed the EPSC evoked by the TA pathway stimulation (Figure 3-1C; 56.8 ± 2.3%,
average of 15 – 20 min after DA application), indicating a decrease in excitatory neurotransmission. We also analyzed paired-pulse facilitation [inversely correlated with vesicle release probability (Katz and Miledi, 1968; Zucker, 1973; Dobrunz and Stevens, 1997)], before and after DA application. After DA application, paired-pulse facilitation was significantly enhanced (Figure 3-1D), suggesting that DA acts, at least in part, via an inhibition of neurotransmitter release. The DA-induced depression was reversible (Figure 3-1E) and blocked by dopamine receptor antagonists (Figure 3-1F).
Figure 3-1: Inhibition of TA-CA1 pyramidal excitatory synaptic transmission by DA (A) Simultaneous extracellular field recording from SC-CA1 and TA-CA1 synapses.
Extracellular recording and stimulating electrodes are placed in stratum lacunosum
moleculare (SLM) or stratum radiatum (SR), respectively. (SO: stratum oriens, SP:
stratum pyramidale). Shown are representative fEPSP recorded in SLM or SR as a result of TA or SC stimulation (scale bar = 0.1 mV, 10 msec). (B) Application of DA (20 µM;
indicated by bar) significantly depressed the fEPSP slope of TA-CA1 synapses, but did not affect SC-CA1 synapses (n = 6) (scale bar = 0.1 mV, 5 msec). (C) Whole-cell patch-clamp recording from CA1 pyramidal neurons in the presence of the GABAA and B
receptor antagonists, bicuculline (10 µM) and CGP 55845A (1 µM). After DA application, the EPSC evoked by TA pathway stimulation was significantly depressed (n = 4) (scale bar = 50 pA, 20 msec) (*p < 0.01). (D) Paired pulse facilitation analysis using the same condition described in C. Pulse interval was 50 msec. Data from individual experiments are represented by small gray circles; large diamonds represent the mean. After DA application, paired pulse facilitation of the TA-CA1 EPSC was significantly enhanced (n
= 5) (scale bar = 20 msec) (*p < 0.01). (E) Recovery from long-term application of DA (n
= 4). (F) Blockade of DA-induced depression at TA-CA1 synapses by dopamine receptor antagonists, SKF 83566 (1 µM) and Sulpiride (10 µM) (DA: n = 8, DA antagonists: n = 5).
3.3.2 DA depresses excitatory synaptic transmission at TA-interneuronal synapses
In addition to excitatory connections with CA1 pyramidal neuron dendrites, the axons of the TA pathway also make synapses with interneurons in area CA1 (Freund and Buzsaki, 1996). Among the various classes of interneurons present, the interneurons located at the border between stratum radiatum and stratum lacunosum-moleculare receive excitatory synapses from the TA pathway. (Lacaille and Schwartzkroin, 1988;
Dvorak-Carbone and Schuman, 1999b). We obtained intracellular recordings from those interneurons and examined the effects of DA on the TA-interneuron excitatory synapse.
We found that DA also depressed the TA-pathway evoked EPSP in interneurons (Figure 3-2B; 57.7 ± 4.2% in EPSP amplitude, 44.0 ± 12.5% in EPSP slope after DA application).
Thus, DA depressed the excitatory synaptic inputs at both TA-pyramidal and TA-interneuron synapses. Although we did not examine connections between TA axons and other type of interneurons in area CA1, considering the presynaptic action of DA (Figure 3-1D), it is possible that other TA-interneuron synapses will be similarly depressed.
Previous studies have reported that relatively strong inhibitory responses can be observed in CA1 pyramidal neurons following TA pathway stimulation (Empson and Heinemann, 1995). Since the TA-CA1 synapses are primarily excitatory (Desmond et al., 1994), the inhibition of pyramidal neurons is caused by interneurons which receive excitatory inputs from the TA pathway and, in turn, make inhibitory connections with pyramidal neurons (Lacaille and Schwartzkroin, 1988). We thus examined the inhibitory
responses in CA1 pyramidal neurons evoked by TA pathway stimulation. Whole-cell monosynaptic IPSCs were recorded from CA1 pyramidal neurons at a holding potential of 0 mV in the presence of glutamate receptor antagonists (CNQX + APV). The monosynaptic IPSC (Figure 3-2D) was not modulated by DA (Figure 3-2E), suggesting that DA does not influence inhibitory synaptic transmission.
Figure 3-2: DA-induced depression of excitatory inputs to SLM interneurons
(A) Intracellular recording from SLM interneurons. Biocytin-filled electrodes were used for the staining of SLM interneurons. The inset shows representative spike activities following current injection (scale bar = 50 msec, 20 mV). (B) After DA application, the EPSP evoked by the TA pathway stimulation was significantly depressed (scale bar = 1
mV, 20 msec) (*p < 0.01). (C) Whole-cell voltage clamp recordings from CA1 pyramidal neurons at a holding potential of 0 mV. The late component of the IPSC disappeared after excitatory blockade with CNQX (10 µM) and APV (25 µM). The size of late IPSC showed large variability among recorded neurons (scale bar = 50 pA, 100 msec). (D) The monosynaptic IPSC was blocked by GABA receptor antagonists, bicuculline (10 µM) and CGP 55845A (1 µM) (scale bar = 50 pA, 100 msec). (E) DA did not influence monosynaptic IPSC (scale bar = 20 pA, 50 msec).
3.3.3 DA-induced presynaptic inhibition enhances synaptic transmission at TA-CA1 synapses during HFS
The above indicate that DA appears to selectively inhibit the excitatory TA connections with both local interneurons and CA1 pyramidal neurons (Figure 3-3A). The DA-induced depression of excitatory inputs to interneurons is predicted to reduce the impact of inhibitory transmission on CA1 pyramidal neurons (disinhibition). Because this inhibitory transmission is disynaptic, the inhibition of pyramidal neurons is delayed relative to excitation. This raises the possibility that DA’s ability to modulate the output of the TA-CA1 circuit may be modulated by stimulation frequency. In particular, we predicted that appropriately-timed TA stimuli would increase the impact of disinhibition (Figure 3-3B).
To test this idea, we examined the net effect of DA during epochs of high-frequency stimulation (HFS). We made extracellular field recordings from the TA-CA1 synapses, before, during and after two epochs of HFS (100 Hz, 100 pulses); the second epoch was delivered after DA application (Figure 3-3C). To quantify the differences between 1st and 2nd HFS, we measured the field potential evoked by the last (100th) stimulus (referred to as the “steady-state potential”). Under control conditions, the steady-state potentials observed at the 1st and 2nd HFS were almost identical (2nd/1st steady-state potential ratio: 0.96 ± 0.04) (Figure 3-3D). DA application, however, significantly enhanced the 2nd steady-state potential (ratio: 1.98 ± 0.13). To examine whether presynaptic inhibition is sufficient to induce this phenomenon, we lowered the extracellular calcium concentration after 1st HFS (low [Ca2+]ext; Figure 3-3C), reducing
the probability of neurotransmitter release. This manipulation mimicked the effect of DA, resulting in a larger steady-state potential at the 2nd HFS (ratio: 1.67 ± 0.15; Figure 3-3D). To exclude the possibility that a smaller fEPSP (induced by DA) may itself induce a larger steady-state potential, independent of release probability, we reduced the stimulus strength to imitate the small fEPSP depressed by DA (reduced stim; Figure 3-3C). This manipulation did not augment the steady-state potential (ratio: 0.60 ± 0.11; Figure 3-3D).
Taken together, these data suggest that presynaptic inhibition induced by DA is responsible for the larger steady-state field potential observed during HFS.
To directly examine the synaptic efficacy of TA-pyramidal neuron synapses, we made whole-cell voltage-clamp recordings from CA1 pyramidal neurons and measured current influx evoked by the TA pathway stimulation in control or DA-treated slices (Figure 3-4A). A comparison of the average TA-elicited EPSC waveforms from control vs. DA-treated slices indicated no apparent differences in the EPSC waveform shape or kinetics (Figure 3-4A, left). Input resistance also did not differ between the groups.
However, during HFS, current influx was significantly larger in the presence of DA (Figure 3-4A, middle and right), suggesting that synaptic efficacy of TA-pyramidal neuron synapses was enhanced by DA. To confirm that the above differences were caused by a modulation of inhibitory transmission, we made recordings under GABAA and B
receptor blockade to isolate excitatory inputs. We found that GABA receptor antagonists completely prevented the facilitation of the steady-state current by DA (Figure 3-4B), indicating that the observed difference (Figure 3-4A) was caused by inhibitory modulation. The above results reinforce the idea that DA-induced disinhibition enhances
synaptic efficacy during high-frequency stimulation.
Figure 3-3: DA augments the steady-state field potential induced by HFS
(A) Scheme of the TA-CA1 synapse. The TA axons make excitatory connections with both pyramidal neurons and interneurons. Interneurons in turn inhibit pyramidal neurons
(feed-forward inhibition). Depression of excitatory inputs onto interneurons by DA reduces inhibition of pyramidal neurons. (B) Frequency dependent effect of feedforward inhibition. In contrast to the direct excitatory TA-CA1 input, the TA pathway exerts a disynaptic modulation of inhibition. Thus, the inhibition of pyramidal neurons after TA stimulation is delayed, relative to the excitation. In pyramidal neurons, the TA pathway-evoked inhibition does not affect excitation during low-frequency stimulation, because of the delay in inhibition. However, during high-frequency stimulation, inhibition can effectively suppress subsequent excitatory responses. (C) Extracellular field recording from TA-CA1 synapses. Left, HFS was applied at 15 min and 45 min (indicated by arrows). Field potential traces during HFS are normalized to the baseline fEPSP amplitude prior to HFS application. The field potential at the end of the HFS (100th stimulus response) was measured (steady-state potential). Right, the average waveforms during HFS are shown. DA: after 1st HFS, DA was applied (indicated by bar).
Low [Ca2+]ext: after 1st HFS, the extracellular calcium concentration was reduced from 2.5 mM to 1.25 mM (indicated by bar). Reduced stim: at 30 min, the stimulation current was reduced to produce a small fEPSP comparable to that observed during DA application (indicated by arrow) (n = 5 for each group). (D) Ratio of steady-state potentials observed during second and first HFS epochs. DA application induced a significantly (*p < 0.01) larger steady-state potential that was also mimicked by reduction of [Ca2+]ext.
Figure 3-4: Enhancement of TA-CA1 synaptic efficacy during HFS via DA-induced disinhibition
(A) Whole-cell voltage clamp recording from CA1 pyramidal neurons. Waveforms represent the average of all data, showing normalized baseline EPSC (left) and current during HFS (middle). There was no difference in the kinetics of EPSC waveforms obtained with or without DA application. Waveforms during HFS (100 Hz, 100 pulses) were normalized to baseline EPSC amplitude prior to HFS, and the current at the end of HFS (100th stimulus response) was measured (steady-state current). The right figure
shows the analysis of steady-state current, showing that DA induced a significantly (*p <
0.01) larger steady-state current. Input resistances were not significantly different (in MΩ); control: 85.8 ± 9.3, DA: 85.0 ± 9.0 (n = 6 for each group) (scale bar = 20 msec).
(B) Same experimental procedure as A under GABA receptor blockade by bicuculline and CGP55845A. GABA blockade attenuated the enhancement of steady-state current by DA. Input resistances were not significantly different (in MΩ); Bic+CGP: 102.0 ± 7.9, Bic+CGP+DA: 98.0 ± 4.6 (n = 5 for each group) (scale bar = 20 msec).
3.3.4 DA imposes a high-pass filter on TA-CA1 synaptic transmission
The oscillatory patterns of neural networks in the mammalian brain cover a wide range of frequencies from approximately 0.05 to 500 Hz (Buzsaki and Draguhn, 2004).
We reasoned that the temporal features of disinhibition may interact with oscillatory dynamics of neural networks to produce frequency-dependent modulatory effects of DA.
To delineate the frequency profile of DA-induced modulation, we stimulated the TA pathway with 100 pulses at frequencies ranging from 5 to 100 Hz (Figure 3-5A and 3-6A).
Under control conditions, steady-state potentials became progressively smaller at stimulation frequencies greater than 10 Hz (Figure 3-6A). On the other hand, under the influence of DA, steady-state potentials were enhanced at all frequencies higher than 10 Hz (Figure 3-6A). Thus, DA exerts a stimulation frequency-dependent modulation of synaptic strength. In another set of analyses, we took into account the DA-induced inhibition of basal transmission by normalizing all potentials to the baseline fEPSP before DA application (Figure 3-5B and 3-6B). Under the influence of DA, steady-state potentials were smaller than control during low-frequency stimulation because of the excitatory depression, however, during high frequency stimulation, the disinhibition overcame the depression and steady-state potentials were larger than control (Figure 3-6B). A similar of results was obtained in a set of experiments conducted at near-physiological temperature (32 – 34 ºC) (Figure 3-6C and 3-6D). These data indicate that the DA-induced disinhibition together with its excitatory depression exhibits an alternate gating of synaptic strength in a frequency-dependent manner – increasing the impact of high-frequency inputs while decreasing the impact of low-frequency signals.
Thus, DA acts as a high-pass filter on TA-CA1 pyramidal neuron signaling.
Figure 3-5: High-pass filtering of TA-CA1 synaptic efficacy by DA
(A) Examination of frequency-dependent modulation by DA, using extracellular field recordings. 100 pulses of different stimulation frequencies, as indicated, were applied.
Data were normalized to the baseline fEPSP amplitude prior to stimulation and each mean fEPSP amplitude during 100-pulse stimulation was plotted (from top to bottom;
control: n = 5, 5, 7, 6, and 5, DA: n = 5, 5, 5, 5, and 5). (B) Same data as in A, but normalized to the baseline fEPSP amplitude before, instead of after, DA application.
Figure 3-6: Analysis of DA-induced filtering at TA-CA1 synapses at room and near-physiological temperatures
(A) Using same data as in figure 3-5, steady-state potentials were measured. In the presence of DA, the steady-state potential became larger during high-frequency stimulation (*p < 0.05 relative to control) (from left to right; control: n = 5, 5, 7, 6, and 5, DA: n = 5, 5, 5, 5, and 5). (B) As in figure 3-5B, data were normalized to baseline fEPSP amplitude prior to DA application. Thus, this figure shows the total effect of DA,
including on the depression of basal synaptic transmission. Under DA application, although the steady-state potential was smaller during low-frequency stimulation, it overcame the depression and became larger than control during high-frequency stimulation (*p < 0.05 relative to control). Note that control error bars are smaller than the symbol size. (C) Similar experiments as A conducted at higher temperature (32 – 34 °C). (D) Similar analysis as B, using data acquired at higher temperature.
3.3.5 DA bi-directionally modulates synaptic plasticity
To assess the functional impact of DA-induced filtering, we next examined the DA’s effect on synaptic plasticity, the sign and strength of which is known to be dependent on stimulation frequency. To test this idea, long-term potentiation (LTP) was induced at TA-CA1 synapses with or without DA (Figure 3-7A). LTP, induced by high-frequency stimulation (5 trains of 100 Hz 100 pulses), was significantly enhanced by DA (control: 126.4 ± 7.2%, DA: 172.6 ± 14.3%, 50 – 60 min after LTP induction), which was blocked by dopamine receptor antagonists (Figure 3-7D and 3-7E). On the other hand, long-term depression (LTD), which is induced by low-frequency stimulation, was attenuated by DA (Figure 3-7B; control: 81.7 ± 3.5%, DA: 94.8 ± 2.3%, 30 – 35 min after LTD induction). Thus, the frequency-dependent signal filtering by DA has a profound functional impact on the magnitude of synaptic plasticity. We next examined whether modulation of inhibitory transmission also underlies this phenomena. Under GABA receptor blockade, LTP at TA-CA1 synapses was enhanced, and DA’s effect on LTP enhancement was occluded (Figure 3-7C; Bic+CGP: 171.5 ± 18.6%, Bic+CGP+DA:
152.1 ± 9.8%, 50 – 60 min after LTP induction), suggesting an obligatory contribution from the inhibitory network.
How quickly can the network adapt to DA signals? Because dopaminergic neurons show both tonic and burst-like activity patterns in vivo (Grace, 1991; Floresco et al., 2003), we examined the sensitivity of this modulation to very brief (10 sec + 1 – 2 min washout) temporally controlled applications of DA (Figure 3-8A and 3-8B). When DA was applied 10 sec before LTP induction, LTP was significantly enhanced when
compared with vehicle applied control (Figure 3-8C; vehicle: 107.0 ± 2.7%, DA: 136.5 ± 8.7%, 50 – 60 min after LTP induction). The application of DA either 3 min before or 10 sec after LTP induction, however, did not enhance TA-CA1 LTP (Figure 3-8D; 3 min before: 111.0 ± 4.0%, 10 sec after: 110.7 ± 5.6%). These data indicate that extremely brief DA application that is coincident with LTP induction is capable of modulating the TA-CA1 plasticity network.
Figure 3-7: DA-induced modulation of synaptic plasticity at TA-CA1 synapses
(A) Enhancement of TA-CA1 LTP by DA. DA was present for the duration of the experiment. Baselinesare normalized to 1.0 in order to examine LTP independent of the depression of basal synaptic transmission by DA. For all traces, the baseline (pre-plasticity) trace is black, the post-plasticity trace is grey (*p < 0.01) (control: n = 7,
DA: n = 8) (scale bar = 0.1 mV, 5 msec). (B) Attenuation of TA-CA1 LTD by DA. Gray bar: LTD induction (*p < 0.01) (control: n = 7, DA: n = 6) (scale bar = 0.1 mV, 5 msec).
(C) Occlusion of DA-induced TA-CA1 LTP enhancement under GABAA and B receptor blockade. Under GABA receptor blockade, DA did not significantly enhance LTP (n = 5 for each group) (scale bar = 0.1 mV, 5 msec). (D) Blockade of DA-induced enhancement of TA-LTP by dopamine receptor antagonists, SKF 83566 (1 µM) and Sulpiride (10 µM) (DA: n = 8, DA antagonists: n = 5). (E) Analysis of LTP at TA-CA1 synapses (50 – 60 min after LTP induction). Dopamine receptor antagonists completely blocked DA-induced enhancement of TA-LTP (control: n = 7, DA: n = 8, DA antagonists: n = 5).
Figure 3-8: Temporally selectivity of LTP enhancement by DA
(A) Acute application of DA. DA was applied for just 10 sec either 10 sec before, 10 sec after, or 3 min before LTP induction. LTP induction protocol was 100 Hz. 100 pulse stimulation, repeated 2 times at an interval of 30 sec. DA was directly applied in the recording chamber for 10 sec and estimated washout time was about 1 – 2 min. (B) fEPSP slopes during acute application of DA. TA pathway was stimulated at 0.5 Hz. DA was directly applied into the recording chamber at 1 min (indicated by arrow) and its effects on synaptic transmission were reversible. (C) DA application at 10 sec before LTP induction significantly enhanced LTP at TA-CA1 synapses (vehicle: n = 7, DA: n = 8).
(D) DA application 3 min before or 10 sec after LTP induction did not enhance LTP (n = 6 for each).
3.3.6 DA-induced disinhibition influences synaptic plasticity at SC-CA1 synapses Although DA-induced excitatory depression is selective to TA-CA1 synapses, the resulting disinhibition may influence another excitatory input to area CA1, the SC pathway. Previous work has shown that TA activity can interfere with LTP induction at SC-CA1 synapses, primarily due to inhibition evoked by the TA pathway stimulation (Levy et al., 1998; Remondes and Schuman, 2002). We reasoned that DA-induced disinhibition may attenuate this LTP interference. We first confirmed that DA had no significant effect on the magnitude of LTP at SC-CA1 synapses up to 1 hr after LTP induction when the SC was stimulated alone either by theta-burst stimulation (TBS) (Figure 3-9C; control: 142.4 ± 5.7%, DA: 139.7 ± 5.3%, 50 – 60 min after LTP induction) or by HFS (Figure 3-9D; control: 179.6 ± 15.5%, DA: 179.5 ± 13.0%). As previously shown, joint TBS applied to both the SC and TA pathways significantly attenuated LTP at SC-CA1 synapses (Figure 3-9E; 112.3 ± 3.0%). To assess the DA-induced disinhibitory effect on LTP interference, the stimulation amplitude was increased after DA application to compensate for the DA-induced excitatory depression (Figure 3-9B). Under this condition, DA significantly attenuated the LTP interference of SC-CA1 synapses elicited by concurrent TA activity (Figure 3-9E; 128.0 ± 1.5%). These results indicate that DA-induced disinhibition has a large impact on plasticity induction not only at TA-CA1 synapses, but also at SC-CA1 synapses.